Papain-induced in vitro disc degeneration model for the study of injectable nucleus pulposus therapy

BACKGROUND CONTEXT Proteolytic enzyme digestion of the intervertebral disc (IVD) offers a method to simulate a condition of disc degeneration for the study of cell-scaffold constructs in the degenerated disc. PURPOSE To characterize an in vitro disc degeneration model (DDM) of different severities of glycosaminoglycans (GAG) and water loss by using papain, and to determine the initial response of the human mesenchymal stem cells (MSCs) introduced into this DDM. STUDY DESIGN Disc degeneration model of a bovine disc explant with an end plate was induced by the injection of papain at various concentrations. Labeled MSCs were later introduced in this model. METHODS Phosphate-buffered saline (PBS control) or papain in various concentrations (3, 15, 30, 60, and 150 U/mL) were injected into the bovine caudal IVD explants. Ten days after the injection, GAG content of the discs was evaluated by dimethylmethylene blue assay and cell viability was determined by live/dead staining together with confocal microscopy. Overall matrix composition was evaluated by histology, and water content was visualized by magnetic resonance imaging. Compressive and torsional stiffness of the DDM were also recorded. In the second part, MSCs were labeled with a fluorescence cell membrane tracker and injected into the nucleus of the DDM or a PBS control. Mesenchymal stem cell viability and distribution were evaluated by confocal microscopy. RESULTS A large drop of GAG and water content of the bovine disc were obtained by injecting >30 U/mL papain. Magnetic resonance imaging showed Grade II, III, and IV disc degeneration by injecting 30, 60, and 150 U/mL papain. A cavity in the center of the disc could facilitate later injection of the nucleus pulposus tissue engineering construct while retaining an intact annulus fibrosus. The remaining disc cell viability was not affected. Mesenchymal stem cells injected into the protease-treated DDM disc showed significantly higher cell viability than when injected into the PBS-injected control disc. CONCLUSIONS By varying the concentration of papain for injection, an increasing amount of GAG and water loss could be induced to simulate the different severities of disc degeneration. MSC suspension introduced into the disc has a very low short-term survival. However, it should be clear that this bovine IVD DDM does not reflect a clinical situation but offers exciting possibilities to test novel tissue engineering protocols.

A Process Model of Complementarity and Substitution of Contractual and Relational Governance in IS Outsourcing

This paper develops a process model of how and why complementarity and substitution form over time between contractual and relational governance in the context of information systems outsourcing. Our analysis identifies four distinct process patterns that explain this formation as the outcome of interaction processes between key elements of both contractual and relational governance. These patterns unveil the dynamic nature of complementarity and substitution. In particular, we show that the relationship between contractual and relational governance oscillates between complementarity and substitution. Those oscillations are triggered mainly by three types of contextual events (goal fuzziness, goal conflict, and goal misalignment). Surprisingly, substitution of informal control did not occur as an immediate reaction to external events but emerged as a consequence of preceding complementarity. Thus, our study challenges the prevailing view of an either/or dichotomy of complementarity and substitution by showing that they are causally connected over time.

Traumatization and chronic pain: a further model of interaction.

Up to 80% of patients with severe posttraumatic stress disorder are suffering from "unexplained" chronic pain. Theories about the links between traumatization and chronic pain have become the subject of increased interest over the last several years. We will give a short summary about the existing interaction models that emphasize particularly psychological and behavioral aspects of this interaction. After a synopsis of the most important psychoneurobiological mechanisms of pain in the context of traumatization, we introduce the hypermnesia-hyperarousal model, which focuses on two psychoneurobiological aspects of the physiology of learning. This hypothesis provides an answer to the hitherto open question about the origin of pain persistence and pain sensitization following a traumatic event and also provides a straightforward explanatory model for educational purposes.

Construct Validity of the Pig Intestine Model in the Simulation of Laparoscopic Urethrovesical Anastomosis

Introduction: Laparoscopic training models are increasingly important in urology to allow trainees to improve their laparoscopic skills prior to going to the operating room. For a training model to be valid, it must correlate with performance in a real case. The model must also discriminate between experienced and inexperienced subjects. [See PDF for complete abstract]

An Emerging Technology: To Model Patient Education for the Dental Hygiene Student

Introduction: Could Livestream Video, an emerging audiovisual media, be used more effectively than the traditional demonstration method of teaching or modeling dental hygiene students on how to provide chairside patient education? [See PDF for complete abstract]

Evaluation and Model of a Clinical Librarian Program

Clinical medical librarianship is entering its second decade, but little evaluative data has accrued in the literature. Variations from the original programs and novel new approaches have insured the survival of the program so far. The clinical librarian (CL) forms a vital link between the library and the health care professional, operating as an important information transfer agent. However, to further insure the survival of these vital programs, hard evaluative evidence is needed. The University of Texas Medical Branch (UTMB) at Galveston began a CL Program in 1978/79. An extensive three-year pre/post evaluation study was conducted using a specifically developed evaluation model, which, if adopted by others, will provide the needed comparative data. Both a pilot study, or formative evaluation, and a summative evaluation were conducted. The results of this evaluation confirmed many of the conclusions reported by other CL programs. Eight hypotheses were proposed at the beginning of this study. Data were collected and used to support acceptance or rejection of the null hypotheses, and conclusions were drawn according to the results. Implications relevant to the study conclusions and future trends in medical librarianship are also discussed in the closing chapter.

Molecular basis of Corynebacterium diphtheriae virulence and infection in the Caenorhabditis elegans model host

Corynebacterium diphtheriae is the causative agent of cutaneous and pharyngeal diphtheria in humans. While lethality is certainly caused by diphtheria toxin, corynebacterial colonization may primarily require proteinaceous fibers called pili, which mediate adherence to specific tissues. The type strain of C. diphtheriae possesses three distinct pilus structures, namely the SpaA, SpaD, and SpaH-type pili, which are encoded by three distinct pilus gene clusters. The pilus is assembled onto the bacterial peptidoglycan by a specific transpeptidase enzyme called sortase. Although the SpaA pili are shown to be specific for pharyngeal cells in vitro, little is known about functions of the three pili in bacterial pathogenesis. This is mainly due to lack of in vivo models of corynebacterial infection. As an alternative to mouse models as mice do not have functional receptors for diphtheria toxin, in this study I use Caenorhabditis elegans as a model host for C. diphtheriae. A simple C. elegans model would be beneficial in determining the specific role of each pilus-type and the literature suggests that C. elegans infection model can be used to study a variety of bacterial species giving insight into bacterial virulence and host-pathogen interactions. My study examines the hypothesis that pili and toxin are major virulent determinants of C. diphtheriae in the C. elegans model host.

Detailed analysis of sequence changes occurring during vlsE antigenic variation in the mouse model of Borrelia burgdorferi infection.

Lyme disease Borrelia can infect humans and animals for months to years, despite the presence of an active host immune response. The vls antigenic variation system, which expresses the surface-exposed lipoprotein VlsE, plays a major role in B. burgdorferi immune evasion. Gene conversion between vls silent cassettes and the vlsE expression site occurs at high frequency during mammalian infection, resulting in sequence variation in the VlsE product. In this study, we examined vlsE sequence variation in B. burgdorferi B31 during mouse infection by analyzing 1,399 clones isolated from bladder, heart, joint, ear, and skin tissues of mice infected for 4 to 365 days. The median number of codon changes increased progressively in C3H/HeN mice from 4 to 28 days post infection, and no clones retained the parental vlsE sequence at 28 days. In contrast, the decrease in the number of clones with the parental vlsE sequence and the increase in the number of sequence changes occurred more gradually in severe combined immunodeficiency (SCID) mice. Clones containing a stop codon were isolated, indicating that continuous expression of full-length VlsE is not required for survival in vivo; also, these clones continued to undergo vlsE recombination. Analysis of clones with apparent single recombination events indicated that recombinations into vlsE are nonselective with regard to the silent cassette utilized, as well as the length and location of the recombination event. Sequence changes as small as one base pair were common. Fifteen percent of recovered vlsE variants contained "template-independent" sequence changes, which clustered in the variable regions of vlsE. We hypothesize that the increased frequency and complexity of vlsE sequence changes observed in clones recovered from immunocompetent mice (as compared with SCID mice) is due to rapid clearance of relatively invariant clones by variable region-specific anti-VlsE antibody responses.

Genetically determined heterogeneity of lung disease in a mouse model of airway mucus obstruction

Mucus clearance is an important airway innate defense mechanism. Airway-targeted overexpression of the epithelial Na(+) channel β-subunit [encoded by sodium channel nonvoltage gated 1, beta subunit (Scnn1b)] in mice [Scnn1b-transgenic (Tg) mice] increases transepithelial Na(+) absorption and dehydrates the airway surface, which produces key features of human obstructive lung diseases, including mucus obstruction, inflammation, and air-space enlargement. Because the first Scnn1b-Tg mice were generated on a mixed background, the impact of genetic background on disease phenotype in Scnn1b-Tg mice is unknown. To explore this issue, congenic Scnn1b-Tg mice strains were generated on C57BL/6N, C3H/HeN, BALB/cJ, and FVB/NJ backgrounds. All strains exhibited a two- to threefold increase in tracheal epithelial Na(+) absorption, and all developed airway mucus obstruction, inflammation, and air-space enlargement. However, there were striking differences in neonatal survival, ranging from 5 to 80% (FVB/NJcontext and timing of airway innate immune dysfunction critically determines lung disease phenotype. These mouse strains may be useful to identify key modifier genes and pathways.

Effects of Combined Bevacizumab and Paclitaxel on Tumor Interstitial Fluid Pressure in a Preclinical Breast Cancer Model

Effects of Combined Bevacizumab and Paclitaxel on Tumor Interstitial Fluid Pressure in a Preclinical Breast Cancer Model by Ricardo H. Alvarez Several mechanisms of cell resistance are often accountable for unsuccessful chemotherapy against cancer. Another reason, which has received increased attention, is the inefficient transport of anticancer drugs into tumor tissue. These impaired transports of chemotherapy into the tumor have been attributed to abnormal microvasculature and to pathologically increased tumor hypertension also called: interstitial fluid pressure (IFP). The pathophysiological processes leading to elevated tumor IFP are poorly understood. Here, in a preclinical breast cancer model, it is argued that a condition of raised IFP is a major factor in preventing optimal access of systemically administered chemotherapy agents. In our experimental model, we used a GILM2 human breast cancer in xenografts; mice were treated with different doses of paclitaxel –a widely used antimicrotubular agent, and bevacizumab –monoclonal antibody against vascular endothelial growth factor (VEGF). The proposed research project is designed to test the hypothesis that paclitaxel in combination with bevacizumab decreases the tumor IPF by restoring tumor permeability and increasing chemotherapy delivery. We demonstrated that the combination of paclitaxel and bevacizumab produced greater tumor control than either agent given alone and this combination reduced the IFP, producing an increment of 75% of apoptosis compared with the control arm. In addition, the intra-tumor paclitaxel quantification by liquid chromatography/Mass Spectrometry (LC/MS) demonstrated that lower dose of both agents showed a synergistic effect compared with high dose of treatment, where there is no significantly increase of paclitaxel into the tumor. These preclinical results are likely to have broad implications for the utility of anti-angiogenic therapies alone and in combination with chemotherapeutic agents.

ROLE OF VITAMIN-D3 AND RETINOIC ACID IN A HUMAN THP-1 MACROPHAGE MODEL OF MYCOBACTERIUM TUBERCULOSIS INFECTION

Mycobacterium tuberculosis (Mtb) replicates within the human macrophages and we investigated the activating effects of retinoic acid (RA) and vitamin D3 (VD) on macrophages in relation to the viability of Mtb. A combination of these vitamins (RAVD) enhanced the receptors on THP-1 macrophage (Mannose receptor and DC-SIGN) that increased mycobacterial uptake but inhibited thesubsequent intracellular growth of Mtb by inducing reactive oxygen species (ROS) and autophagy. RAVD also enhanced antigen presenting and homing receptors in THPs that suggested an activated phenotype for THPs following RAVD treatment. RAVD mediated activation was also associated with a marked phenotypic change in Mtb infected THPs that fused with adjacent cells to formmultinucleate giant cells (MNGCs). Typically MNGCs occurred over 30 days of in vitro culture and contained non-replicating persisting Mtb for as long as 60 days in culture. We propose that the RAVD mediated inhibition of replicating Mtb leading to persistence of non-replicating Mtb within THPs may provide a novel human macrophage model simulating formation of MNGCs in humanlungs.

From school to work: A comprehensive model of work-related motivation in the transition to working life.

Motivation is a core concept to understand work related outcomes and vocational pursuits. However, existing research mostly focused on specific aspects of motivation, such as goals or self-efficacy beliefs, while falling short of adequately addressing more complex and integrative notions of motivation. Advancing the current state of research, we draw from Motivational Systems Theory and a model of proactive motivation to propose a comprehensive model of work-related motivation. Specifically, we define motivation as a system of mutually related factors consisting of goals, emotions, and personal agency beliefs, comprised by capability beliefs and context evaluations. Adapting this model of motivation to the school-to-work transition, we postulate that this motivational system is affected by different social, personal, and environmental variables, for example social support, the presence of role-models, personality traits, and scholastic achievement. We further expect that students with more autonomous work-related goals, expectations of more positive emotional experiences in their future working life, fewer perceived barriers to their career development, and higher work-related self-efficacy beliefs would be more successful in their transition from school to work. We also propose that goal-directed engagement acts as a partial mediator in the relationship between motivation and a successful transition. Finally, we hypothesize that work-related motivation while in school will have meaningful effects on positive outcomes while in vocational training, as represented by more work engagement, higher career commitment, job satisfaction, and lower intentions to quit training. In sum, we advance the point that the adaptation of a broader concept of work-related motivation in the school-to-work transition would result in more powerful predictions of success in this transition and would enhance scientific research and interventions in career development and counselling practice.

Asking Sensitive Questions in Online Surveys: An Experimental Comparison of the Randomized Response Technique and the Crosswise Model

Self-administered online surveys provide a higher level of privacy protection to respondents than surveys administered by an interviewer. Yet, studies show that asking sensitive questions is problematic also in self-administered mode. Because respondents might not be willing to reveal the truth and provide answers that are subject to social desirability bias, the validity of prevalence estimates of sensitive behaviors gained via online surveys can be challenged. A well-known method to combat these problems is the Randomized Response Technique (RRT). However, convincing evidence that the RRT provides more valid estimates than direct questioning in online mode is still lacking. Moreover, an alternative approach called the Crosswise Model (CM) has recently been suggested to overcome some of the deficiencies of the RRT. In the context of an online survey on plagiarism and cheating on exams among students of two Swiss universities (N = 6,494), we tested different implementations of the RRT and the CM and compared them to direct questioning using a randomized experimental design. Results reveal a poor performance of the RRT, which failed to elicit higher prevalence estimates than direct questioning. Using the CM however, significantly higher prevalence estimates were obtained making it a promising new alternative to the conventional RRT.

A model of late long-term potentiation simulates aspects of memory maintenance.

Late long-term potentiation (L-LTP) denotes long-lasting strengthening of synapses between neurons. L-LTP appears essential for the formation of long-term memory, with memories at least partly encoded by patterns of strengthened synapses. How memories are preserved for months or years, despite molecular turnover, is not well understood. Ongoing recurrent neuronal activity, during memory recall or during sleep, has been hypothesized to preferentially potentiate strong synapses, preserving memories. This hypothesis has not been evaluated in the context of a mathematical model representing ongoing activity and biochemical pathways important for L-LTP. In this study, ongoing activity was incorporated into two such models - a reduced model that represents some of the essential biochemical processes, and a more detailed published model. The reduced model represents synaptic tagging and gene induction simply and intuitively, and the detailed model adds activation of essential kinases by Ca(2+). Ongoing activity was modeled as continual brief elevations of Ca(2+). In each model, two stable states of synaptic strength/weight resulted. Positive feedback between synaptic weight and the amplitude of ongoing Ca(2+) transients underlies this bistability. A tetanic or theta-burst stimulus switches a model synapse from a low basal weight to a high weight that is stabilized by ongoing activity. Bistability was robust to parameter variations in both models. Simulations illustrated that prolonged periods of decreased activity reset synaptic strengths to low values, suggesting a plausible forgetting mechanism. However, episodic activity with shorter inactive intervals maintained strong synapses. Both models support experimental predictions. Tests of these predictions are expected to further understanding of how neuronal activity is coupled to maintenance of synaptic strength. Further investigations that examine the dynamics of activity and synaptic maintenance can be expected to help in understanding how memories are preserved for up to a lifetime in animals including humans.

Uptake And Metabolism Of 5’-AMP In The Erythrocyte Play Key Roles In The 5’-AMP Induced Model Of Deep Hypometabolism

UPTAKE AND METABOLISM OF 5’-AMP IN THE ERYTHROCYTE PLAY KEY ROLES IN THE 5’-AMP INDUCED MODEL OF DEEP HYPOMETABOLISM Publication No. ________ Isadora Susan Daniels, B.A. Supervisory Professor: Cheng Chi Lee, Ph.D. Mechanisms that initiate and control the natural hypometabolic states of mammals are poorly understood. The laboratory developed a model of deep hypometabolism (DH) initiated by uptake of 5’-adenosine monophosphate (5’-AMP) into erythrocytes. Mice enter DH when given a high dose of 5’-AMP and the body cools readily. Influx of 5’-AMP appears to inhibit thermoregulatory control. In a 15°C environment, mice injected with 5’-AMP (0.5 mg/gw) enter a Phase I response in which oxygen consumption (VO2) drops rapidly to 1/3rd of euthermic levels. The Phase I response appears independent of body temperature (Tb). This is followed by gradual body temperature decline that correlates with VO2 decline, called Phase II response. Within 90 minutes, mouse Tb approaches 15°C, and VO2 is 1/10th of normal. Mice can remain several hours in this state, before gradually and safely recovering. The DH state translates to other mammalian species. Our studies show uptake and metabolism of 5’-AMP in erythrocytes causes biochemical changes that initiate DH. Increased AMP shifts the adenylate equilibrium toward ADP formation, consequently decreasing intracellular ATP. In turn, glycolysis slows, indicated by increased glucose and decreased lactate. 2,3-bisphosphoglycerate levels rise, allosterically reducing oxygen affinity for hemoglobin, and deoxyhemoglobin rises. Less oxygen transport to tissues likely triggers the DH model. The major intracellular pathway for AMP catabolism is catalyzed by AMP deaminase (AMPD). Multiple AMPD isozymes are expressed in various tissues, but erythrocytes only have AMPD3. Mice lacking AMPD3 were created to study control of the DH model, specifically in erythrocytes. Telemetric measurements demonstrate lower Tb and difficulty maintaining Tb under moderate metabolic stress. A more dramatic response to lower dose of 5’-AMP suggests AMPD activity in the erythrocyte plays an important role in control of the DH model. Analysis of adenylates in erythrocyte lysate shows 3-fold higher levels of ATP and ADP but similar AMP levels to wild-type. Taken together, results indicate alterations in energy status of erythrocytes can induce a hypometabolic state. AMPD3 control of AMP catabolism is important in controlling the DH model. Genetically reducing AMP catabolism in erythrocytes causes a phenotype of lower Tb and compromised ability to maintain temperature homeostasis.